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Antibodies (also known as immunoglobulins, abbreviated Ig) are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They are typically made of basic structural units—each with two large heavy chains and two small light chains—to form, for example, monomers with one unit, dimers with two units or pentamers with five units. Antibodies are produced by a kind of white blood cell called a B cell. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.
Although the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures to exist. This region is known as the hypervariable region. Each of these variants can bind to a different target, known as an antigen. This huge diversity of antibodies allows the immune system to recognize an equally wide diversity of antigens. The unique part of the antigen recognized by an antibody is called an epitope. These epitopes bind with their antibody in a highly specific interaction, called induced fit, that allows antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules that make up an organism. Recognition of an antigen by an antibody tags it for attack by other parts of the immune system. Antibodies can also neutralize targets directly by, for example, binding to a part of a pathogen that it needs to cause an infection.
The large and diverse population of antibodies is generated by random combinations of a set of gene segments that encode different antigen binding sites (or paratopes), followed by random mutations in this area of the antibody gene, which create further diversity. Antibody genes also re-organize in a process called class switching that changes the base of the heavy chain to another, creating a different isotype of the antibody that retains the antigen specific variable region. This allows a single antibody to be used by several different parts of the immune system. Production of antibodies is the main function of the humoral immune system.
Forms
Antibodies occur in two forms: a soluble form secreted into the blood and other fluids in the body, and a membrane-bound form that is attached to the surface of a B cell. Soluble antibodies that are secreted from an activated B cell (in its plasma cell form) bind to foreign substances and signal for their destruction by the rest of the immune system. They may also be called free antibodies (until they bind an antigen and become part of an immune complex) or secreted antibodies.
The membrane-bound form of an antibody may be called a surface immunoglobulin (sIg) or a membrane immunoglobulin (mIg). It is part of the B cell receptor (BCR), which allows a B cell to detect when a specific antigen is present in the body and triggers B cell activation. The BCR is composed of surface-bound IgD or IgM antibodies and associated Ig-? and Ig-? heterodimers, which are capable of signal transduction. A typical human B cell will have 50,000 to 100,000 antibodies bound to its surface. Upon antigen binding, they cluster in large patches, which can exceed 1 micrometer in diameter, on lipid rafts that isolate the BCRs from most other cell signaling receptors. These patches may improve the efficiency of the cellular immune response. In humans, the cell surface is bare around the B cell receptors for several thousand Ångstroms, which further isolates the BCRs from competing influences.
Isotypes
Antibodies can come in different varieties known as isotypes or classes. In placental mammals there are five antibody isotypes known as IgA, IgD, IgE,IgG and IgM. They are each named with an "Ig" prefix that stands for immunoglobulin, another name for antibody, and differ in their biological properties, functional locations and ability to deal with different antigens, as depicted in the table.
The antibody isotype of a B cell changes during cell development and activation. Immature B cells, which have never been exposed to an antigen, are known as naïve B cells and express only the IgM isotype in a cell surface bound form. B cells begin to express both IgM and IgD when they reach maturity—the co-expression of both these immunoglobulin isotypes renders the B cell 'mature' and ready to respond to antigen. B cell activation follows engagement of the cell bound antibody molecule with an antigen, causing the cell to divide and differentiate into an antibody producing cell called a plasma cell. In this activated form, the B cell starts to produce antibody in a secreted form rather than a membrane-bound form. Some daughter cells of the activated B cells undergo isotype switching, a mechanism that causes the production of antibodies to change from IgM or IgD to the other antibody isotypes, IgE, IgA or IgG, that have defined roles in the immune system.
Structure
Antibodies are heavy (~150kDa) globular plasma proteins that are also known as immunoglobulins. They have sugar chains added to some of their amino acid residues. In other words, antibodies are glycoproteins. The basic functional unit of each antibody is an immunoglobulin (Ig) monomer (containing only one Ig unit); secreted antibodies can also be dimeric with two Ig units as with IgA, tetrameric with four Ig units like teleost fish IgM, or pentameric with five Ig units, like mammalian IgM.
Immunoglobulin domains
The Ig monomer is a "Y"-shaped molecule that consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds. Each chain is composed of structural domains called Ig domains. These domains contain about 70-110 amino acids and are classified into different categories (for example, variable or IgV, and constant or IgC) according to their size and function. They have a characteristic immunoglobulin fold in which two beta sheets create a “sandwich” shape, held together by interactions between conserved cysteines and other charged amino acids.
Heavy chain
There are five types of mammalian Ig heavy chain denoted by the Greek letters: α, δ, ε, γ, and μ. The type of heavy chain present defines the class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Distinct heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids.
Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant region composed of three tandem (in a line) Ig domains, and a hinge region for added flexibility; heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain.
Light chain
In mammals there are two types of light chain, which are called lambda (λ) and kappa (κ). A light chain has two successive domains: one constant domain and one variable domain. The approximate length of a light chain is 211 to 217 amino acids. Each antibody contains two light chains that are always identical; only one type of light chain, κ or λ, is present per antibody in mammals. Other types of light chains, such as the iota (ι) chain, are found in lower vertebrates like Chondrichthyes and Teleostei.
CDRs, Fv, Fab and Fc Regions
Some parts of an antibody have unique functions. The tips of the Y, for example, contain the site that bind antigen and, therefore, recognize specific foreign objects. This region of the antibody is called the Fab (fragment, antigen binding) region. It is composed of one constant and one variable domain from each heavy and light chain of the antibody. The paratope is shaped at the amino terminal end of the antibody monomer by the variable domains from the heavy and light chains. The variable domain is also referred to as the FV region and is the most important region for binding to antigens. More specifically variable loops, three each on the light (VL) and heavy (VH) chains are responsible for binding to the antigen. These loops are referred to as the Complementarity Determining Regions (CDRs).
The base of the Y plays a role in modulating immune cell activity. This region is called the Fc (Fragment, crystallizable) region, and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils.
Research applications
Specific antibodies are produced by injecting an antigen into a mammal, such as a mouse, rat or rabbit for small quantities of antibody, or goat, sheep, or horse for large quantities of antibody. Blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen—in the serum, which can now be called antiserum. Antigens are also injected into chickens for generation of polyclonal antibodies in egg yolk. To obtain antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies. Polyclonal and monoclonal antibodies are often purified using Protein A/G or antigen-affinity chromatography
In research, purified antibodies are used in many applications. They are most commonly used to identify and locate intracellular and extracellular proteins. Antibodies are used in flow cytometry to differentiate cell types by the proteins they express; different types of cell express different combinations of cluster of differentiation molecules on their surface, and produce different intracellular and secretable proteins. They are also used in immunoprecipitation to separate proteins and anything bound to them (co-immunoprecipitation) from other molecules in a cell lysate, in Western blot analyses to identify proteins separated by electrophoresis, and in immunohistochemistry or immunofluorescence to examine protein expression in tissue sections or to locate proteins within cells with the assistance of a microscope. Proteins can also be detected and quantified with antibodies, using ELISA and ELISPOT techniques.
Primary antibodies
Primary antibodies are antibodies raised against an antigenic target of interest (a protein, peptide, carbohydrate, or other small molecule) and are typically unconjugated (unlabelled).Primary antibodies that recognize and bind with high affinity and specificity to unique epitopes across a broad spectrum of biomolecules are available as high specificity monoclonal antibodies and/or as polyclonal antibodies. These antibodies are useful not only to detect specific biomolecules but also to measure changes in their level and specificity of modification by processes such as phosphorylation, methylation, or glycosylation. A primary antibody can be very useful for the detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease and they are used for the study of ADME and multi-drug resistance (MDR) of therapeutic agents.
Secondary antibodies
A secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications.
Secondary antibodies may be polyclonal or monoclonal, and are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions.
Specific secondary antibodies are usually chosen to work in specific laboratory applications. Frequently, any one of several secondary antibodies perform adequately in a particular application. They are selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred.